转座子插入诱变捕获团头鲂耐低氧主控基因的研究

DNA transposons are transposable elements which can move within their host genomes by changing their insertional positions in a process called transposition. The mutagenesis strategy mediated by transposon insertion will be extremely powerful tools for trapping the target genes that control important trait in fish species. The present study will construct suitable transposon insertional mutagenesis system for Megalobrama amblycephala, based on a new cyprinid hAT family transposon termed Tgf2, which has recently been discovered in goldfish (Carassius auratus) in our lab. Firstly, the Tgf2 transposase and transposon double positive strain of Megalobrama amblycephala will be constructed by co-injected plasmids Tgf2[SA-RFP]DS with prm1-TPase. Next, efficient transposon insertion mutant libraries of Megalobrama amblycephala will be constructed through mating the double transgenic male with wild-type female, as a result of transposase can induce efficient transpose in germ cells. Then, the hypoxia tolerance candidate mutants will be screened by hypoxia stress in the transposon insertion mutant libraries. And forward genetic approach is used to further identify hypoxia tolerance mutants of Megalobrama amblycephala. Finally, the target genes controlling hypoxia tolerance as well as their function will be identified in hypoxia tolerance mutants of Megalobrama amblycephala using reverse PCR, chromosome FISH and transposon precisely cut-off and traits recovery method. The aim of this study is to explore a novel method for efficient insertion mutagenesis in Megalobrama amblycephala genome and to rapidly identificate the target genes that control hypoxia tolerance. The present study expresses an innovated idea, which will promote the molecular breeding for hypoxia-tolerant trait in Megalobrama amblycephala in future.

DNA转座子(Transposon)可通过高效转座，插入生物基因组特定基因的内部或邻近位点，获得相关性状插入诱变突变体。基于转座子的插入诱变策略在鱼类重要性状主控基因的筛选、功能解释方面有着重要的研究前景。本项目拟在近年我们发现的一个新的鲤科鱼类hAT家族转座子，即金鱼Tgf2转座子的基础上，构建Tgf2转座酶和转座子双转基因团头鲂系；利用转座酶可诱导转座子在雄配子中高效转座的特性，通过双转基因团头鲂雄亲本与野生型雌亲本的交配，建立团头鲂Tgf2转座子高效插入突变体库；通过低氧胁迫，筛选出团头鲂耐低氧候选突变体，进而采用正向遗传学方法，甄别出团头鲂耐低氧突变体；通过染色体定位和转座切离-性状回复研究，鉴定出团头鲂耐低氧主控基因或元件及功能模式。本研究旨在探索一种开展团头鲂基因组高效插入诱变的新方法，并快速获得团头鲂耐低氧主控基因，以促进团头鲂耐低氧分子育种，在研究思路上具有较大的创新性。

自2012年执行以来，严格按照合约计划执行，并认真开展研究工作。主要的研究进展为：（1）构建了N末端不同长度的Tgf2转座酶蛋白，通过诱导转座酶的可溶性原核表达和液相色谱纯化，获得了在鱼类细胞中具有高活性的L-Tgf2TPase转座酶蛋白系统，解决了采用Tgf2转座酶mRNA介导鱼类基因组转座过程中出现的效率低、操作难的制约；（2）构建了Tgf2-SB双转座子基因捕获插入诱变系统1个，进行了团头鲂插入诱变研究，建立了一个Tgf2转座子介导的团头鲂插入诱变P0库，包含团头鲂耐低氧性状相关的显性二龄突变体160尾，具有明显的耐低氧性能；（3）获得与团头鲂耐低氧性状通路密切相关的功能基因或基因型4个，克隆了团头鲂低氧应答相关的IGFBP-1（胰岛素样生长因子结合蛋白1）和EPO（促红细胞生成素）基因，获得了团头鲂Ctgfa/b重复基因cDNA的全长序列，鉴定出团头鲂egln1a/1b、egln2与egln3基因，鉴定出团头鲂vhl、hif1an与vegfaa基因，并进行了这些基因在低氧胁迫下的功能研究。（4）针对团头鲂不耐低氧问题，在团头鲂“浦江1号”基础上通过家系选育和低氧胁迫选育出F3代耐低氧新品系，为获得团头鲂“浦江2号”新品种奠定了基础。（5）通过转录组学分析、小RNA组学分析、蛋白组学分析、功能基因研究等探索团头鲂新品系的耐低氧分子机制，为后续选育提供理论指导。迄今共发表论文10篇，其中SCI论文8篇，核心期刊论文2篇；授权国家发明专利2项；实审国家发明专利1项；PCT国际专利1项（已进入美国）；培养研究生5名。